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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Streszczenie

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 °C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

Wprowadzenie

Serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) are the most abundant protein in plasma and play a vital role in maintaining the osmotic pressure of the blood compartment. They are also recognized as carrier proteins for small molecules of low water solubility, such as steroids, fatty acids, thyroid hormones, and a wide variety of drugs. The binding property (e.g., binding sites, binding affinity or strength) of these molecules to serum albumins forms an important topic in pharmacokinetics.1-4 Several analytical methods have been developed to study the binding properties of different drugs to serum albumins, such as X-ray crystallography,5,6 nuclear magnetic resonance (NMR),7-11 and surface plasmon resonance (SPR),12,13 etc. However, these methods are constrained by either a tedious and time-consuming analyzing process (e.g., growth of single crystal for X-ray crystallographic study), requirement of specialized and expensive equipment (SPR), or in need of costly isotope labeling (NMR) for detection. It is therefore highly desirable to develop alternative ways for small molecular drug screening in a fast, straight-forward, and cost-efficient manner.

Gold nanoclusters (Au NCs) are a special type of nanomaterial, which contain several to tens of metal atoms with sizes smaller than 2 nm.14-17 They have attracted extensive research interests due to their discrete and size-dependent electronic structure,18,19 and molecular-like absorptions and emissions.20-23 Such unique materials properties, in particular the strong fluorescence, have found diverse applications such as sensing and imaging in biological systems. 24-32 Ultrasmall fluorescent Au NCs can be synthesized using functional proteins, such as serum albumins, as template. 33 In a typical protein-templated synthesis of Au NCs, a certain amount of Au salts are first encapsulated inside the protein and subsequently reduced by the protein itself. The reducing ability of the protein is attributed to constituent functional amino acid residues (e.g., tyrosine) that can be activated by increasing the solution pH to alkaline. Unfolding of protein structure is considered as a critical step for the formation of Au NCs. This is because in an unfolded protein, more reducing functional groups can be exposed to the encapsulated Au salts. Protein unfolding can be achieved by heat treatment or exposure to denaturing agents. Introduction of small molecular drugs can also affect the unfolding process, i.e. modifying the midpoint denaturation temperature and the enthalpy of unfolding. 34,35 The effect of all these factors, in turn can be reflected by the formation kinetics of fluorescent Au NCs and manifested in the fluorescence intensity of resultant Au NCs.36

This video demonstrates the method of drug screening by synthesizing Au NCs in drug-loaded albumin proteins at a higher temperature (60 °C) or in the presence of denaturing agents (e.g., urea). The fluorescence intensity of resultant Au NCs is the signal readout. First, Au NCs are synthesized in HSA and BSA templates treated at 60 °C or in the presence of urea to show how protein unfolding (induced by heat treatment or denaturants) affects the formation kinetics of Au NCs. Second, Au NCs are synthesized in protein templates preloaded with different drugs, and the drug loading effect on the relative fluorescence intensities of resultant Au NCs is studied, which provide the measure of relative binding strength. Finally, the Au NC-drug screening protocol is modified for quantitative measurement of drug-protein binding constant (KD) by varying the drug content preloaded in the protein of a fixed concentration.

Protokół

Caution: Please consult the safety data sheets (SDS) of all involved chemicals before use. The drug screening experiment involves the synthesis and handling of nanomaterials, which may have additional hazards compared to their bulk counterpart. Please ensure all necessary control measures to be practiced throughout the experiment, including the use of engineering controls (fume hood) and personal protective equipment (PPE, e.g., safety length pants, closed-toe shoes, chemical resistant gloves, and safety goggles).

1. Preparation of Chemical Reagents for Drug Screening

  1. Precursors for Au NCs synthesis
    1. Dissolve 30 mg of gold (III) chloride solution (99.99% trace metals basis, 30 wt.% in dilute HCl) in 6.9 ml of ultrapure water to prepare 15 mM of gold (III) chloride solution. Caution: gold chloride solution is corrosive and irritant. Wear proper PPE to avoid direct contact with eye and skin.
    2. Dissolve 74 mg of HSA or BSA in 1 ml of ultrapure water to prepare 74 mg/ml of protein stock solution.
  2. Drug solutions
    1. Dissolve the desired amount of drug, e.g., 1.9 mg for (a) ibuprofen, 2.8 mg for (b) warfarin, 2.3 mg for (c) phenytoin, and 1.5 mg for (d) sulfanilamide in 20 µl of DMSO to prepare 450 mM of drug stock solutions.
      Note: DMSO is selected as the solvent because these drugs are hydrophobic and have poor solubility in water.
  3. Other reagents
    1. Dissolve 600 mg of NaOH pellets in 10 ml of ultrapure water to prepare 1.5 M of NaOH solution. Dissolve 2.4 g of urea in 2 ml of ultrapure water to prepare 20 M of urea solution.

2. Synthesis of Protein-Templated Au NCs

  1. HSA-templated Au NCs (HSA-Au NCs)
    1. Place two glass vials, each containing a micro magnetic stir bar in it, on two separate temperature controllable magnetic stirrers.
    2. Set the temperature of one magnetic stirrer to 60 °C, and label the glass vial on top of it as “60 °C”; while the other magnetic stirrer is kept at room temperature (RT) where the glass vial on top of it is labeled as “RT”.
    3. Add 200 µl of HSA solution, 200 µl of ultrapure water, and 200 µl of gold (III) chloride solution to each vial under constant stirring (set as 360 rpm unless otherwise specified) to allow the encapsulation of Au ions inside the protein template.
    4. 2 min later, add 20 µl of NaOH solution to each vial so as to activate the reducing capability of HSA in forming Au NCs and start to record the reaction time as 0 min.
    5. Every 20 min, draw 50 µl of solutions from each of the sample to a 384-well black plate and measure the emission spectrum with a microplate reader. The typical scanning setup: λex = 370 nm, λem = 410 – 850 nm.
    6. Stop the magnetic stirrer after 100 min.
    7. Cool down the glass vials under running water in a sink. Caution: The glass vials are hot. Wear heat resistant gloves to avoid burning.
    8. Plot the photoemission spectra at all time for each sample to acquire the formation kinetics of Au NCs at different temperature conditions.
  2. BSA-templated Au NCs (BSA-Au NCs)
    1. Place a glass vial containing a micro magnetic stir bar on the top of a magnetic stirrer. Leave the temperature as room temperature.
    2. Mix 200 µl of BSA solution, 200 µl of urea, and 200 µl of gold (III) chloride solution in the glass vial under constant stirring.
    3. 2 min later, add 20 µl of NaOH solution to activate the reducing capability of BSA to form Au NCs and start to record the reaction time as 0 min. Measure the photoemission spectrum of the reaction mixture hourly.
      Note: The photoemission spectrum of BSA-Au NCs is measured less frequently because the formation rate of BSA-Au NCs is slower than that of HSA-Au at the same temperature due to less effectiveness of urea to unfold the protein.
    4. Stop the magnetic stirrer after 7 hr. Plot the photoemission spectra at all time to see the formation kinetics of Au NCs by urea denaturation.

3. Small Molecular Drug Screening

  1. Screen relative binding affinity of different small molecular drugs to HSA
    1. Place five glass vials, each containing a magnetic stir bar in it, on top of a temperature controllable multipoint magnetic stirrer. Label these vials as a, b, c and d respectively for each drug, namely ibuprofen, warfarin, phenytoin, and sulfanilamide. Label the one with pure HSA as control.
    2. Add 200 µl of HSA to each vial. Add 1 µl of drug solution to the four corresponding vials. Add 1 µl of DMSO to the control. Switch on the stirrer and set the spin speed to 360 rpm and incubate for 1 hr to allow the drug binding to HSA to complete.
    3. 1 hr later, add 200 µl of Milli-Q water and 200 µl of gold (III) chloride solution to each vial under constant stirring. Set the temperature to 60 °C under constant stirring for 10 min. Add 20 µl of 1.5 M NaOH to each vial and start to record the reaction time as 0 min.
    4. Quickly draw 50 µl of solution from each vial to a 384-well black plate and measure the emission spectrum (results labeled as 0 min). Record the emission spectra of each sample every 10 min. Collect at least four spectra at four different time, that is 0, 10, 20, and 30 min.
    5. Repeat steps 3.1.1 - 3.1.4 several times to obtain results with a consistent trend. Stop the magnetic stirrer.
    6. Plot the time-resolved photoemission spectra for each sample (a - d, and control). Identify the peak intensity of all samples and plot against the time to compare the formation kinetics of Au NCs in different drug-loaded protein templates.
  2. Measure the binding constant of a specific drug to HSA
    1. Repeat steps 3.1.1 – 3.1.4. Replace the drug solution with ibuprofen solutions at four different concentrations (including a control sample using HSA only without drug loading). Label the glass vial according to the drug concentration correspondingly.
    2. 10 min later, cool down the glass vials under running water in a sink. Caution: The glass vials are hot. Wear heat resistant gloves to avoid burning.
    3. Draw 50 µl of the above solutions from each vial to a 384-well black plate and measure the emission spectrum.
    4. Repeat steps 3.2.1 – 3.2.3 twice more to obtain another two sets of results at different drug concentrations.
    5. Stop the magnetic stirrer. Plot the photoemission spectra and analyze the results.
      1. For the raw data obtained in each individual batch, plot the photoemission spectra of each sample in software such as OriginPro software and find out the peak intensity.
      2. Calculate and plot the relative fluorescence intensity [(I0 - I) /I0] against the drug concentration, where I and I0 refer to the fluorescence intensity of Au NCs formed in drug-loaded HSA and pure HSA, respectively.
      3. Calculate the binding constant by fitting the data to a single site binding model using the Michaelis-Menten equation: Y = Rmax × C/(KD + C), where Rmax indicates the maximum response signal, C is the concentration of ligand and KD is the binding constant. Perform this in software such as OriginPro. Select the menu Analysis Fitting Nonlinear Curve Fit, then select Hill function from Growth/Sigmoidal category. On the Settings: Function Selection page, click Fit to show up the fitted results.

Wyniki

Protein unfolding is an important procedure for the formation of protein-templated Au NCs because more reactive functional groups (e.g., tyrosine residues) of a protein can be exposed to reduce the encapsulated Au ions and thus accelerate the formation rate of Au NCs. Heating and external denaturing agents are two common means to promote the protein unfolding process. Figure 1 demonstrates the effect of heating and adding external denaturing agents on the formation kinetics of Au NCs, using HSA ...

Dyskusje

There are several critical steps that need to be highlighted in this method. In the protocol of screening the relative binding affinity of different small molecular drugs, Steps 3.1.2, 3.1.3, and 3.1.4 are critical to obtain good results showing consistent trend for the relative binding strength. In these steps, the actions of adding chemicals and drawing reaction solutions for measurement should be as quickly as possible to minimize the time lag effect and the same sequence of adding chemicals and drawing reaction solut...

Ujawnienia

The authors have nothing to disclose.

Podziękowania

Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Materiały

NameCompanyCatalog NumberComments
Gold (III) chloride solution, 30%Sigma-Aldrich484385Corrosive, irritant
Human serum albumin, 96%Sigma-AldrichA1887
Bovine Serum albumin, 96%Sigma-AldrichA2153
Ibuprofen, 98%Sigma-AldrichI4883 
warfarin, 98%Sigma-AldrichA2250
phenytoinSigma-AldrichPHR1139
sulphanilamide, 99%Sigma-AldrichS9251
dimethyl sulfoxideSigma-AldrichD8418
ureaSigma-AldrichU5128
Sodium hydroxideSigma-Aldrich221465
Magnetic stirrerIKART5
Microplate readerTecanInfinite M200
384-well plateCorning
5 ml air displacement pipetteEppendorf
1,000 μl air displacement pipetteEppendorf
100 μl air displacement pipetteEppendorf
5,000 μl Eppendorf tips
1,000 μl Eppendorf tips
100 μl Eppendorf tips
1.5 ml micro tubeEppendorf
20 ml glass vial with screw cap
4 ml glass vial with screw cap

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Fluorimetric MethodProtein targetingSmall Molecule Drug ScreeningFluorescent Gold NanoclustersAlbumin ProteinsDrug protein Binding AffinityDrug protein Binding Constant

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